Iron is known to be an essential element of the makeup of every living organism, but may also become toxic at physiological pH values by virtue of its tending to oxidize, hydrolyze and precipitate as insoluble ferric oxide polymers. The protein ferritin, found in all living cells, is the body's means for ensuring that iron toxicity does not occur. Ferritin functions by storing iron in the cells in a soluble and readily available form. The iron stored in cells may then be mobilized whenever needed by the body, for example for erythropoiesis.
The name “ferritin” actually encompasses a number of individual isomeric forms which are characteristic of different tissue types. Each isoferritin has 24 subunits of two distinct types, being light subunits (L) and heavy subunits (H). These subunits differ in molecular weight, the light subunit being about 18 kDa, and the heavy subunit about 19–21 kDa. The isoferritins extracted from different tissues or organs typically exhibit different isoelectric points, with the isoelectric focusing pattern of human tissues forming a continuous spectrum: those tissues associated with high iron storage have ferritin at the basic end of the spectrum (e.g. spleen and liver), while iron poor tissues, (e.g. heart and placenta) and malignant cells have acidic ferritin. (Drysdale, J., Ciba Found. Symp., 51:41, 1976). The difference in isoelectric point appears to be related to the different distribution of light and heavy subunits in each type. Specifically, heavy subunits-rich ferritin are relatively acidic, and light chain rich ferritin are relatively basic (Covell, et al., in Ferritin and Isoferritins as Biochemical Markers, p. 49–65, 1984, Elsevier). Current studies indicate that the H and L subunits are encoded by a complex group of genes.
A specific type of acidic isoferritin has been shown to be characteristic of neoplastic cells and placental cells (Drysdale and Singer, Cancer Res., 34:3352, 1974). This protein is also known as oncofetal ferritin or placental isoferritin (PLF). Human placental ferritin has been shown to be composed predominantly of a single subunit type comigrating with a liver ferritin standard on SDS-PAGE (Brown et al., Biochem. J., 182:763, 1979). However, an immunoradiometric assay performed with anti-human spleen ferritin has shown tissue specific antigenicity for PLF (Brown et al., supra). A three subunit structure has been revealed for PLF (Moroz et al., G. I. Pat. Clin., 1:17–23, 1986). In addition to the L and H subunits characteristic of all ferritin, there is also a high molecular weight (43 kDa) subunit which appears to be unique for human placenta, and thus provides a potential site for identification of the placental isoferritin molecule as distinguished from any other type of ferritin.
Various ferritin isoforms have been isolated from normal and malignant tissues, the most acidic ones predominating in tumor and fetal tissues (Drysdale J., 1976, Ciba Found. Symp. 51:41; Arosio et al., J. Biol. Chem., 253:4451, 1978). It has been suggested that the assay or acidic isoferritin in the serum may be of value in the diagnosis of malignancy (Hazard et al., Nature, 265:755, 1977). Elevated concentrations of serum ferritin were found in patients suffering from a variety of malignant diseases, including acute lymphocytic leukemia (ALL) (Matzner et al., Am. J. Hematol., 9:13, 1980), hepatoma (Giannoulis, Digestion, 30:236, 1984) and recently Hodgkin's disease (Bezwoda et al., Scand. J. Haematol., 35:505, 1985). In assays based on antibodies HeLa cell ferritin, Hazard and Drysdale found higher concentrations of ferritin in sera from patients with various tumors than in the same sera assayed by antibodies directed against normal liver ferritin (Hazard et al., supra). Others have failed to demonstrate a consistent pattern of isoferritins in tumor tissues (Cragg et al., Br. J. Cancer, 35:635, 1977; Halliday et al., Cancer Res., 36:4486, 1976) or in sera obtained from patients with tumors (Jones et al., Clin. Chim. Acta., 85:81, 1978; Jones et al., Clin. Chim. Acta., 106:203, 1980).
Although publications concerning the existence of oncofetal ferritin or placental isoferritins have been evident at least since the year 1976, up until today the sequence of this protein and the gene encoding therefor were not known. This is probably due to the fact that the protein itself is hydrophobic and almost devoid of iron and as a consequence extremely sticky and not capable of sedimentation even by high speed centrifugation thus hindering its isolation and purification. In addition, the sequence of the gene coding for oncofetal ferritin could not be found in regular cDNA libraries, probably due to the fact that its expression in these libraries is extremely low. The protein is secreted only by the placenta during pregnancy or by cancer cells in malignant diseases such as lymphoproliferative disorders, breast cancer and in HIV infection.
Breast cancer is a malignant disease effecting different populations at a rate of one to every 9–13 of women. Early diagnosis of breast cancer is known to considerably improve the prognosis of the patient. Diagnosis of breast cancer is based today mainly on imagining techniques such as mamma graphs verified at times by biopsies. Blood-based assays of breast cancer have been reported in the literature, for example, biomarker such as CA 15.3 (Daly, L. et al., Comparison of a novel assay for breast cancer mucin to and CA 54 15.3 carcinoembryonic antigen, J. Clin. Oncol., 10:1057–65, 1992); the CA 549(2) marker (Dormers, I. J., et al., CA 549; a new tumor marker for patients with advanced breast cancer J. Clin. Lab. Anal., 2:168–73, 1988); and the marker CA M29 CEA (Duistrian, A. M. et al., Evaluation of CA M26, CA M29, CA 15.3 and CEA as circulating tumor markers in breast cancer patients. Tumor Biol., 12:82–90, 1991). However these assays, reported in the scientific community have not gained, to date, clinical significance (Werner M., et al., Clinical utility and validation of emerging biochemical markers from mammary adenocarcinoma, Clin. Chem., 39/11(B):2386–96, 1993).
U.S. Pat. No. 4,882,270 discloses an assay for the detection of breast cancer based on determination of oncofetal ferritin. The assay is based on binding of the oncofetal ferritin to specific monoclonal antibodies.
Pathological pregnancy is a term commonly used to describe a multitude of symptoms which create difficulties in carrying a child to term and include spontaneous abortion and miscarriage, premature contractions, toxemia, premature delivery. U.S. Pat. No. 4,954,434 discloses the fact that low levels or absence of PLF in pregnant women can serve as a marker for potentially high risk pregnancy. Detection of this state is again achieved by monoclonal antibodies which has PLF specificity. This patent also concerns treatment and prevention of actual and potentially pathological pregnancy by the administration of this protein. However, since the sequence of the protein was not known at the date of the patents, the treatment suggested involved administration of partially purified protein and not of recombinant pure proteins.
U.S. Pat. Nos. 5,571,678, 5,120,640 and 5,283,177 are all directed to methods for assaying the presence and evaluating the prognosis of acquired immunodeficiency associated with HIV induction, by determining levels of placental isoferritin by monoclonal antibodies.
All the above detection methods concern antibody-based assays. While such assays are known to be useful in conditions where the level of the protein to be detected is quite high, they are notorious for eliciting a false-negative answer where the protein level is low. Against this, assays based on amplification of mRNA (RT-PCR) are much more sensitive and can detect even minute expression of mRNA. Thus there is need today for a RT-PCR method for detection of oncofetal ferritin for detection of breast cancer and for diagnosis of high risk pregnancies at its early stage.
Furthermore, it would have been desirable to provide pure oncofetal ferritin protein prepared by recombinant processes for therapeutic and vaccination purposes.